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Chromatin structure remodeling (RSC) complex
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<h2 id="rsc-vs-swisnf">RSC vs. SWI/SNF</h2> <p>While RSC and SWI/SNF have similar structures and functions, RSC is significantly more common than the <a href="/facts/SWI%2fSNF/cxssxQVq">SWI/SNF</a> complex in yeast, and RSC is required for <a href="/facts/Mitosis/ExxrSUs8">mitotic cell division</a>.<a class="footnote-ref" id="fnref:5" href="#fn:5"><sup>5</sup></a> Absence of the RSC complex is lethal; without it, cells cannot survive.<a class="footnote-ref" id="fnref:6" href="#fn:6"><sup>6</sup></a> RSC consists of a complex of 15 <a href="/facts/Protein_subunit/HhuET9UP">protein subunits</a>, and at least three of these subunits are conserved between RSC and <a href="/facts/SWI%2fSNF/cxssxQVq">SWI/SNF</a>.<a class="footnote-ref" id="fnref:7" href="#fn:7"><sup>7</sup></a> RSC and SWI/SNF are composed of very similar components, such as the Sth1 components in RSC and the SWI2/Snf2p in SWI/SNF. Both of these components are <a href="/facts/ATPase/8aEJlNgl">ATPases</a> that consist of Arp7 and Arp9, proteins similar to <a href="/facts/Actin/Hu3qIUL9">actin</a>.<a class="footnote-ref" id="fnref:8" href="#fn:8"><sup>8</sup></a> The subunits of Sth1 (Rsc6p, Rsc8p, and Sfh1p) are <a href="/facts/Paralogues/mCIVStrA">paralogues</a> to the three subunits of SWI/SNF (Swp73p, Swi3p, and Snf5p). While there are many similarities between these two chromatin remodeling complexes, they remodel different parts of chromatin.<a class="footnote-ref" id="fnref:9" href="#fn:9"><sup>9</sup></a> They also have opposing roles, specifically when interacting with the <i>PHO8</i> <a href="/facts/Promoter_(genetics)/YDYBzLfg">promoter</a>. RSC works to guarantee the placement of nucleosome N-3, while SWI/SNF attempts to override the placement of N-3.<a class="footnote-ref" id="fnref:10" href="#fn:10"><sup>10</sup></a> </p><p>RSC and SWI/SNF complexes both function as chromatin remodeling complexes in humans (<i><a href="/facts/Homo_sapiens/rlcTENsr">Homo sapiens</a></i>) and the common fruit fly (<i><a href="/facts/Drosophila_melanogaster/KtnHVCcB">Drosophila melanogaster</a></i>). SWI/SNF was first discovered when a genetic screen was done in yeast with a mutation causing a deficiency in mating-type switching (swi) and a mutation causing a deficiency in sucrose fermentation.<a class="footnote-ref" id="fnref:11" href="#fn:11"><sup>11</sup></a> After this chromatin remodeling complex was discovered, the RSC complex was found when its components, Snf2 and Swi2p, were discovered to be homologous to the SWI/SNF complex. </p><p><a href="/facts/BLAST_(biotechnology)/uWufGxFH">BLAST</a> homology alignment of reference genomes has revealed that the yeast RSC complex is even more similar to the human SWI/SNF complex than it is to yeast's own SWI/SNF complex.<a class="footnote-ref" id="fnref:12" href="#fn:12"><sup>12</sup></a> </p> <h2 id="the-role-of-rsc">The role of RSC</h2> <p>The role of nucleosomes is a very important topic of study in modern <a href="/facts/Epigenetics/UfAAERcn">epigenetics</a> research. It is known that nucleosomes interfere with the binding of transcription factors to DNA, therefore allowing them some form of subtle control over the probability of transcription and replication occurring at a given locus. With the help of an <i>in vitro</i> experiment using yeast, it was discovered that RSC is required for nucleosome remodeling. There is evidence that RSC does not remodel the nucleosomes on its own; it uses information from enzymes to help position nucleosomes. </p><p>The <a href="/facts/ATPase/8aEJlNgl">ATPase</a> activity of the RSC complex is activated by single-stranded, double-stranded, and/or nucleosomal DNA, while some of the other chromatin remodeling complexes are only stimulated by one of these DNA-types.<a class="footnote-ref" id="fnref:13" href="#fn:13"><sup>13</sup></a> </p><p>The RSC complex (specifically Rsc8 and Rsc30) is crucial when fixing double-stranded breaks via <a href="/facts/Non-homologous_end_joining/xvHqwBPt">non-homologous end joining</a> (NHEJ) in yeast.<a class="footnote-ref" id="fnref:14" href="#fn:14"><sup>14</sup></a> This repair mechanism is important for cell survival as well as for maintaining an organism's genome. These double-stranded breaks are typically caused by radiation, and they can be detrimental to the genome. The breaks can lead to mutations that reposition a chromosome and can even lead to the entire loss of a chromosome. The mutations associated with double-stranded breaks have been linked to cancer and other deadly genetic diseases.<a class="footnote-ref" id="fnref:15" href="#fn:15"><sup>15</sup></a> RSC not only repairs double-stranded breaks by <a href="/facts/Non-homologous_end_joining/xvHqwBPt">NHEJ</a>, it also repairs these breaks using <a href="/facts/Homologous_recombination/WU0IIVVv">homologous recombination</a> with the help of the SWI/SNF complex.<a class="footnote-ref" id="fnref:16" href="#fn:16"><sup>16</sup></a> SWI/SNF is recruited first, prior to two homologous chromosomes bind, and then RSC is recruited to help complete the repair.<a class="footnote-ref" id="fnref:17" href="#fn:17"><sup>17</sup></a> </p> <h2 id="mechanism-of-action-in-dsdna">Mechanism of action in dsDNA</h2> <p>A single-molecule study using <a href="/facts/Magnetic_tweezers/WpJnq8AX">magnetic tweezers</a> and linear DNA observed that RSC generates DNA loops <i>in vitro</i> while simultaneously generating negative <a href="/facts/DNA_supercoil/vFTgbyNT">supercoils</a> in the template.<a class="footnote-ref" id="fnref:18" href="#fn:18"><sup>18</sup></a> These loops can consist of hundreds of base pairs, but the length depends on how tightly the DNA is wound, as well as how much ATP is present during this translocation.<a class="footnote-ref" id="fnref:19" href="#fn:19"><sup>19</sup></a> Not only could RSC generate loops, but it was also able to relax these loops, meaning that the translocation of RSC is reversible.<a class="footnote-ref" id="fnref:20" href="#fn:20"><sup>20</sup></a> </p><p>Hydrolysis of <a href="/facts/Adenosine_triphosphate/nB9bwgcU">ATP</a> allows the complex to translocate the DNA into a loop. RSC can release the loop either by translocating back to the original state at a comparable velocity, or by losing one of its two contacts.<a class="footnote-ref" id="fnref:21" href="#fn:21"><sup>21</sup></a> </p> <h2 id="rsc-components">RSC components</h2> <p>The following is a list of RSC components that have been identified in yeast, their corresponding human <a href="/facts/Ortholog/mCIVStrA">orthologs</a>, and their functions: </p> <table><tbody><tr><th>Yeast</th><th>Human</th><th>Function</th></tr><tr><td><i><a href="https://www.yeastgenome.org/locus/RSC1">RSC1</a></i></td><td>BAF180</td><td>DNA repair mechanisms, <a href="/facts/Tumor_suppressor/Awte4S51">tumor suppressor</a> protein <a class="footnote-ref" id="fnref:22" href="#fn:22"><sup>22</sup></a></td></tr><tr><td><i><a href="https://www.yeastgenome.org/locus/RSC2">RSC2</a></i></td><td>BAF180</td><td>DNA repair mechanisms, tumor suppressor protein <a class="footnote-ref" id="fnref:23" href="#fn:23"><sup>23</sup></a></td></tr><tr><td><i><a href="https://www.yeastgenome.org/locus/RSC4">RSC4</a></i></td><td>BAF180</td><td>DNA repair mechanisms, tumor suppressor protein <a class="footnote-ref" id="fnref:24" href="#fn:24"><sup>24</sup></a></td></tr><tr><td><i><a href="https://www.yeastgenome.org/locus/RSC6">RSC6</a></i></td><td>BAF60a</td><td><a href="/facts/Mitosis/ExxrSUs8">Mitotic</a> growth<a class="footnote-ref" id="fnref:25" href="#fn:25"><sup>25</sup></a></td></tr><tr><td><i><a href="https://www.yeastgenome.org/locus/RSC8">RSC8</a></i></td><td>BAF170, BAF155</td><td>Regulates <a href="/facts/Cerebral_cortex/GpXosGHP">cortical</a> size/thickness,<a class="footnote-ref" id="fnref:26" href="#fn:26"><sup>26</sup></a> tumor suppressor <a class="footnote-ref" id="fnref:27" href="#fn:27"><sup>27</sup></a></td></tr></tbody></table> <h2 id="see-also">See also</h2> <ul><li><a href="/facts/SWI%2fSNF/cxssxQVq">SWI/SNF</a> (nucleosome remodeling complex)</li> <li><a href="/facts/Mi-2%2fNuRD_complex/gY2Vly2Y">Mi-2/NuRD complex</a></li> <li><a href="/facts/INO80B_(gene)/ukzMk01j">INO80B (gene)</a></li> <li><a href="/facts/Imitation_SWI/pYLvBrrJ">Imitation SWI</a> (nucleosome remodeling complex)</li></ul> <h2 id="external-links">External links</h2> <ul><li><a href="https://web.archive.org/web/20090803083240/http://www.scivee.tv/node/5532">Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation</a></li></ul> <h2 id="references">References</h2> <ol> <li id="fn:1"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:1" class="footnote-back-ref">↩</a></p></li> <li id="fn:2"><p>Clapier CR, Iwasa J, Cairns BR, Peterson CL (July 2017). "Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes". Nature Reviews. Molecular Cell Biology. 18 (7): 407–422. doi:10.1038/nrm.2017.26. PMC 8127953. PMID 28512350. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127953" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127953</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></p></li> <li id="fn:3"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></p></li> <li id="fn:4"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:4" class="footnote-back-ref">↩</a></p></li> <li id="fn:5"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:5" class="footnote-back-ref">↩</a></p></li> <li id="fn:6"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></p></li> <li id="fn:7"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:7" class="footnote-back-ref">↩</a></p></li> <li id="fn:8"><p>Tang L, Nogales E, Ciferri C (June 2010). "Structure and function of SWI/SNF chromatin remodeling complexes and mechanistic implications for transcription". Progress in Biophysics and Molecular Biology. 102 (2–3): 122–8. doi:10.1016/j.pbiomolbio.2010.05.001. PMC 2924208. PMID 20493208. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924208" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924208</a> <a href="#fnref:8" class="footnote-back-ref">↩</a></p></li> <li id="fn:9"><p>Tang L, Nogales E, Ciferri C (June 2010). "Structure and function of SWI/SNF chromatin remodeling complexes and mechanistic implications for transcription". Progress in Biophysics and Molecular Biology. 102 (2–3): 122–8. doi:10.1016/j.pbiomolbio.2010.05.001. PMC 2924208. PMID 20493208. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924208" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924208</a> <a href="#fnref:9" class="footnote-back-ref">↩</a></p></li> <li id="fn:10"><p>Smith CL, Horowitz-Scherer R, Flanagan JF, Woodcock CL, Peterson CL (February 2003). "Structural analysis of the yeast SWI/SNF chromatin remodeling complex". Nature Structural Biology. 10 (2): 141–5. doi:10.1038/nsb888. PMID 12524530. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:10" class="footnote-back-ref">↩</a></p></li> <li id="fn:11"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:11" class="footnote-back-ref">↩</a></p></li> <li id="fn:12"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:12" class="footnote-back-ref">↩</a></p></li> <li id="fn:13"><p>Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, Erdjument-Bromage H, et al. (December 1996). "RSC, an essential, abundant chromatin-remodeling complex". Cell. 87 (7): 1249–60. doi:10.1016/S0092-8674(00)81820-6. PMID 8980231. <a href="https://doi.org/10.1016%2FS0092-8674%2800%2981820-6" target="_blank">https://doi.org/10.1016%2FS0092-8674%2800%2981820-6</a> <a href="#fnref:13" class="footnote-back-ref">↩</a></p></li> <li id="fn:14"><p>Shim EY, Ma JL, Oum JH, Yanez Y, Lee SE (May 2005). "The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks". Molecular and Cellular Biology. 25 (10): 3934–44. doi:10.1128/mcb.25.10.3934-3944.2005. PMC 1087737. PMID 15870268. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087737" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087737</a> <a href="#fnref:14" class="footnote-back-ref">↩</a></p></li> <li id="fn:15"><p>Shim EY, Ma JL, Oum JH, Yanez Y, Lee SE (May 2005). "The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks". Molecular and Cellular Biology. 25 (10): 3934–44. doi:10.1128/mcb.25.10.3934-3944.2005. PMC 1087737. PMID 15870268. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087737" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087737</a> <a href="#fnref:15" class="footnote-back-ref">↩</a></p></li> <li id="fn:16"><p>Chai B, Huang J, Cairns BR, Laurent BC (July 2005). "Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair". Genes & Development. 19 (14): 1656–61. doi:10.1101/gad.1273105. PMC 1176001. PMID 16024655. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176001" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176001</a> <a href="#fnref:16" class="footnote-back-ref">↩</a></p></li> <li id="fn:17"><p>Chai B, Huang J, Cairns BR, Laurent BC (July 2005). "Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair". Genes & Development. 19 (14): 1656–61. doi:10.1101/gad.1273105. PMC 1176001. PMID 16024655. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176001" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176001</a> <a href="#fnref:17" class="footnote-back-ref">↩</a></p></li> <li id="fn:18"><p>Lia G, Praly E, Ferreira H, Stockdale C, Tse-Dinh YC, Dunlap D, et al. (February 2006). "Direct observation of DNA distortion by the RSC complex". Molecular Cell. 21 (3): 417–25. doi:10.1016/j.molcel.2005.12.013. PMC 3443744. PMID 16455496. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744</a> <a href="#fnref:18" class="footnote-back-ref">↩</a></p></li> <li id="fn:19"><p>Lia G, Praly E, Ferreira H, Stockdale C, Tse-Dinh YC, Dunlap D, et al. (February 2006). "Direct observation of DNA distortion by the RSC complex". Molecular Cell. 21 (3): 417–25. doi:10.1016/j.molcel.2005.12.013. PMC 3443744. PMID 16455496. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744</a> <a href="#fnref:19" class="footnote-back-ref">↩</a></p></li> <li id="fn:20"><p>Lia G, Praly E, Ferreira H, Stockdale C, Tse-Dinh YC, Dunlap D, et al. (February 2006). "Direct observation of DNA distortion by the RSC complex". Molecular Cell. 21 (3): 417–25. doi:10.1016/j.molcel.2005.12.013. PMC 3443744. PMID 16455496. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744</a> <a href="#fnref:20" class="footnote-back-ref">↩</a></p></li> <li id="fn:21"><p>Lia G, Praly E, Ferreira H, Stockdale C, Tse-Dinh YC, Dunlap D, et al. (February 2006). "Direct observation of DNA distortion by the RSC complex". Molecular Cell. 21 (3): 417–25. doi:10.1016/j.molcel.2005.12.013. PMC 3443744. PMID 16455496. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443744</a> <a href="#fnref:21" class="footnote-back-ref">↩</a></p></li> <li id="fn:22"><p>Hopson S, Thompson MJ (October 2017). "BAF180: Its Roles in DNA Repair and Consequences in Cancer". ACS Chemical Biology. 12 (10): 2482–2490. doi:10.1021/acschembio.7b00541. PMID 28921948. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:22" class="footnote-back-ref">↩</a></p></li> <li id="fn:23"><p>Hopson S, Thompson MJ (October 2017). "BAF180: Its Roles in DNA Repair and Consequences in Cancer". ACS Chemical Biology. 12 (10): 2482–2490. doi:10.1021/acschembio.7b00541. PMID 28921948. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:23" class="footnote-back-ref">↩</a></p></li> <li id="fn:24"><p>Hopson S, Thompson MJ (October 2017). "BAF180: Its Roles in DNA Repair and Consequences in Cancer". ACS Chemical Biology. 12 (10): 2482–2490. doi:10.1021/acschembio.7b00541. PMID 28921948. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:24" class="footnote-back-ref">↩</a></p></li> <li id="fn:25"><p>"RSC6 | SGD". www.yeastgenome.org. Retrieved 2020-03-31. <a href="https://www.yeastgenome.org/locus/S000000648" target="_blank">https://www.yeastgenome.org/locus/S000000648</a> <a href="#fnref:25" class="footnote-back-ref">↩</a></p></li> <li id="fn:26"><p>Tuoc TC, Boretius S, Sansom SN, Pitulescu ME, Frahm J, Livesey FJ, Stoykova A (May 2013). "Chromatin regulation by BAF170 controls cerebral cortical size and thickness". Developmental Cell. 25 (3): 256–69. doi:10.1016/j.devcel.2013.04.005. hdl:11858/00-001M-0000-0013-F327-3. PMID 23643363. <a href="https://doi.org/10.1016%2Fj.devcel.2013.04.005" target="_blank">https://doi.org/10.1016%2Fj.devcel.2013.04.005</a> <a href="#fnref:26" class="footnote-back-ref">↩</a></p></li> <li id="fn:27"><p>DelBove J, Rosson G, Strobeck M, Chen J, Archer TK, Wang W, et al. (December 2011). "Identification of a core member of the SWI/SNF complex, BAF155/SMARCC1, as a human tumor suppressor gene". Epigenetics. 6 (12): 1444–53. doi:10.4161/epi.6.12.18492. PMC 3256333. PMID 22139574. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256333" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256333</a> <a href="#fnref:27" class="footnote-back-ref">↩</a></p></li> </ol>